Virus purification



tion of Indiana Filed Mar. 29, 1965, Ser No.

No Drawing.

Claims. (Cl. 167- 78) assess in The present invention relates to thepurification and concentration of virus materials. More particularly, itrelates to an improved method for the separation of viruses and virusvaccines from dilute and/or crude preparations thereof, whereby the saidvirus materials are obtained in concentrated and stabilized form.

The propagation of viruses takes place only within growing cells, withthe result that virus preparations are contaminated with culture mediamaterials and cell debris, including associated proteinaceoussubstances. Such materials are undesirable in vaccines, and for thisreason a variety of methods have been devised to purity and concentratevirus cultures and derivatives. method, developed by Salk (Proc. Soc.Exp. Biol. Med, 46 (1941), 70 9, 712) and further studied by Stanley(Science, 101 (1945), 332-635), adsorbed virus from an impuretissue-containing broth by in situ precipitation of calcium phosphate,then separated the virus-containing adsorbate, dissolved the calciumphopshate at around pH 6.5 with aqueous sodium citrate solution, anddialyzcd to remove salts.

The present invention is an improvement in the method of Salk, and moreparticularly in the method for the recovery of the virus materials fromthe calcium phosphate adsorbate.

It is thus an object of the invention to improve the recovery of virusmaterials from cultures thereof. Another object is to separate virusmaterials from proteinaceous contaminants. Another object is to prepareviruses in increased concentration over the levels at which they occurin culture thereof. Another object is to prepare virus vaccines of highpotency, high antigenicity, improved stability, and minimal anaphylacticpotential. Other objects will be apparent from the followingdescription.

In accordance with the invention, an adsorbate of virus on in situprecipitated calcium phosphate is dispersed in an aqueous solution ofethylenediaminetetraacetic acid (EDTA), pH 7.88.3, preferably pH8.0-8.2, whereby the calcium phosphate in the adsorbate is convertedinto a complex with the EDTA and is dispersed in the aqueous medium,thereby liberating suiting virus-containing solution is dialyzed againstwater or preferably against aqueous glycine-sodium chloride solution toreduce the content of phosphate and EDTA as desired, the residualphosphate being useful as a buffering agent, and the residual EDTAserving the important function of stabilizing the virus material. Thepurified virus-containing solution is further prepared in whatever formis appropriate for its intended use.

In one embodiment of the invention, a formalin-inacti- Virus Materialthe virus material. The rey United States Patent Ofilice Zi,ild,l53Patented Apr. 25, lQd? vated poliomyelitis vaccine comprising a balancedmixture of the three strains of polio virus at a total concentration ofapproximately 10 -10 virus particles per milliliter is treated accordingto the following procedure. the vaccine is added aqueous 1 M Na HPOstock solution to a concentration of 0.02 l M CaCl stock solution to0.012 M, yielding a tate of calcium hydrogen orthophosphate dihydrate.The resulting slurry is stirred for about 10' minutes, then placed in achill room for 2 hours at about 5 C. The solid phase is separated bycentrifugation. The solid thus obtained is dispersed in an about 0.75 Maqueous solution of ethylenediaminetetraacetic acid (pH adjusted toaround 7.8-8.3 with conc. NaOH), whereby the solid phase is dissolvedand the calcium phosphate-virus adsorbate is caused to dissociate. Theresulting virus-containing solution is dialyzed against aqueous 0.25 Mglycine-0.75 M sodium chloride solution to reduce theethylenediaminetetraacetic acid level below about 6.0025 M, calculatedto the virus dilution ultimately to be employed for injection.

The virus material employed in the present invention can be a virus, anattenuated virus, or an inactivated virus, produced in any of thevarious ways known to the art. Viruses are conveniently grown in fertileeggs or in aqueous liquids containing egg constituents or tissue culturegrowth media, as the case may be. The viruses may be attenuated byexposure to radiation or to the controlled action of chemicaldeactivating agents, or by repeated passage through one or moredifferent types of tissue cultures. inactivated viruses are commonly obavirus material to the action of formaldehyde or other chemicaldeactivating agent under conditions effective to prevent further growthof the virus. They may be preserved, both before and after treatmentaccording to the invention, by adding thimerosal thereto to aconcentration around 120,000.

The process of the invention is broadly applicable to the purificationand concentration of viruses, and finds its principal utility in thetreatment of pathogenic viruses. Illustrative examples of such virusesinclude the causative organisms of measles, influenzaQparainfiuenza,

The calcium phopshate adsorption step employed in conjunction with thepresent invention is carried out by metathetical reaction of suitableprecursor salts in situ in the virus-containing liquid; e.g., by thereaction of calcium chloride, calcium nitrate, calcium. acetate, or thelike with the sodium orthophosphates, potassium 0rthophosphates, or thelike. It is convenient to add the prevanous types of viruses, theoptimum proportion of reactants tends to vary, the follow- 60 ing beingexemplary:

Type

Polio virus Polio vaccine, inactivated ifluenza vaccine Parainfiuenzavaccine Mum ps vaccine. Measels vaccine- The d-isproportionation of thevirus-calcium phosphate idsorbate in accordance with the presentinvention is car- .i6d out with an aqueous solution ofe-thylenediaminetetraicetic acid (EDTA, edathamil, edetic acid) of aconcentration between about 0.1 M and about 1 M and a pH between 7.8 and8.3, higher concentrations being effective to give higher concentrationsof virus, and the optimum pH level being between 8.0 and 8.2. The EDTAis conveniently employed in the form of the sodium, potassium, ammonium,or other nontoxic water-soluble salt, the ratio of cation to anion beingadjusted to produce a pH of the desired level. The quantity of EDTAsolution can vary considerably, according to the amount of calciumphosphate to be dissolved, but generally runs from about 10 to about 50ml. per liter of the original virus-containing liquid. Obvious.y, thesmaller the amount of EDTA solution employed, the more concentrated willbe the resulting vinus solution.

The purified virus-containing liquid obtained in accordance with thepresent invention is optionally adsorbed upon an adjuvant solid havingpharmaceutically acceptable properties upon injection, the proportion ofsolid being preferably sufficient to adsorb substantially all of theantigenic virus therefrom. The virus is further concentrated in this wayand is greatly increased in antigenicity. A preferred solid is hydratedaluminum phosphate, used in a proportion between about 0.5 and aboutmilligrams per milliliter or somewhat higher, according to the virusconcentration. The resulting adsorbed vaccine can be used as such, orcan be sedimented and decanted to yield a product of further increasedpotency. The vaccine, whether thus treated or not, can be furthertreated in a known manner to improve its stability by addition of sodiumbisulfite, sodium sulfamate, glycine, protein hydrolyzates, or the like,or in general any substance which contains an aldehyde-reactive aminoradical or an aldehyde-reactive sulfi-te radical. The material isfinally subjected to the usual tests for antigenicity, contamination,and identity, and is dispensed into ampoules or vaccine bottles forstorage and distribution.

The invention will be more fully understood from the following operatingexamples, which are submitted as illustrative only and not by way oflimitation.

Example 1 The following procedure illustrates the concentration of atrivalent poliornyelitis vaccine, prepared by cultivation of theindividual viruses in monkey kidney cell tissue culture, followed byinactivation with formaldehyde according to the Salk procedure, andpooling in equipotent proportion to produce a trivalent vaccinecontaining around polio virus particles per milliliter. In the example,the following assay procedures were used. Polio virus activity assayswere done using primary monkey kidney cells, or in some cases MK cellsin a plaque system, substantially equivalent results being obtained byboth methods. Protein assays were carried out by the method of Low-ry etal., J. 'Biol. Chem., 193 (November 1951), page 256, or by semimicroKjeldahl, the results again being comparable.

To 18 liters of the trivalent poliomyelitis vaccine contained in aS-gallon bottle were added, with constant stirring, 180 ml. of 1 Mdibasic sodium phosphate solution, followed by 270 ml. of 1 M calciumchloride solution. Stirring was continued thereafter for 20 minutes at asufficient intensity to keep the precipitate of calcium orthophosphatedihydra-te in suspension. The resulting slurry was placed in a chillroom at approximately 5 C. for 24 hours to allow the precipitate tosettle. The supernatant fluid was then withdrawn and discarded. Theprecipitates from 3 bottles were pooled in a l2-gallon bottle andslur-ried therein with around 10 gallons of distilled Water, thenallowed to settle for 48 hours. The supernatant fluid was withdrawn anddiscarded. To the solids were slowly added approximately 360 ml. ofaqueous 0.7 5 M ethylenediaminetetraacetic acid, pH 8.0 (sodiumhydroxide), with rapid stirring, the precipitate being dissolvedthereby. To the concentrated fluid vaccine obtained in this way wasadded polysorbitan monooleate, 20 mcgn/rnk, and the mix ture was thendialyzed for approximately 72 hours against an aqueous solution ofglycine (0.25 M), sodium chloride (0.75 M), and polysorbitan monooleate(20 meg/ml.) to remove the calcium-ethylenediaminetetraacetic acidcomplex, dialysis being continued until the ethylenediaminetetraace-ticacid concentration had reached a level equiva lent to about 0.0 125 Mupon dilution of the vaccine for injection.

The results of the cbove procedure were as follows.

Monkey Potency* Concen- Dose,

*Ratio to antigenicity of NIH reference vaccine.

Example 2 A batch of type I inactivated poliomyelitis vaccine wassubjected to purification generally according to the procedure ofExample 1, and was found to be increased in virus concentration by afactor of approximately 12.9:1. The concentrated vaccine was found tohave undergone a purification of percent, as indicated by its decreasein protein nitrogen assay. Furthermore, it was found to have improvedgreatly in stability, having a chick potency of 1:43 after 42 days at 37C., compared with a complete loss of activity in an untreated controlsample subjected to the same stability test conditions.

Example 3 A 400-liter batch of formalin-inactivated trivalentpoliomyelitis vaccine, prepared from monkey kidney cell tissue culturevirus, was concentrated and purified according to the followingprocedure. The vaccine, contained in a 400-liter tank (rated capacity)equipped with stirrer, was cooled to about 5 C., and to it were addedsuccessively, with constant stirring, sufficient quantities of aqueous 1M sodium dihydrogen phosphate solution and aqueous 1 M calcium chloridesolution to produce a 0.015 M final concentration of each salt. Stirringwas continued for one hour at 5 'C., and the mixture was then allowed tostand at about 5 C. for 2 hours. The precipitate was removed bycentrifuging and dissolved in 7 liters of 0.741 M EDTA solution, pH 8.0,after which polysorbitan monooleate was added to a concentration of 20meg/ml. The solution was dialyzed at 5 C. for 72 hours against anaqueous solution containing 0.25 M glycine, 0.75 M sodium chloride, and20 meg/ml. polysorbitan monooleate, whereby the EDTA concentration wasreduced to approximately 0.006 M. The product contained approximately 10virus particles per milliliter.

Example 4 Influenza virus vaccine, strain Jap. 305, grown in embryonatedeggs, concentrated 10X by centrifuging, and inactivated with formalinaccording to conventional procedure, was further concentrated andpurified according to the procedure and essentially under the conditionsof Example 3. The calcium phosphate precipitate containing the adsorbedvaccine was dissolved in 0.741 M EDTA solution, pH 8.0, employed in theproportion of 35 ml. of solution per liter of the original concentratedvaccine.

The original vaccine, after inactivation, had a chick cell agglutinin(CCA) titer of 822 'CCA units per milliliter. By means of the abovetreatment, it was further concentrated 10X, and was found to have atiter of 12,460 CCA units per milliliter. The inactivated vaccineoriginally contained 118 mcg. of protein per CCA units; after treatment,it had 94 *mcg. per 100 units.

- phate in said adsorbate is The treated vaccine was furtherconcentrated and purified by centrifuging through a Charples T-1P viruscentrifuge at a How rate of 550 ml./'hr. and 50,000 r.p.m. The solidswere removed and resuspended in aqueous 0.05 M glycine and 0.15 sodiumchloride solution to a tfinal concentration of 20 above the original x.The treated product contained 60 meg. of protein per 100 CCA units.

Example 5 Parainfluenza virus, types 1, 2, and 3, grown in Primary GreenMonkey tissue culture, and having an infectivity titer of 4.74(expressed as the negative logarithm of the dilution to 'ICID wassuccessfully concentrated and purified according to the procedure andessentially under the conditions of Example 1. The treated virus, afterdialysis, was found to have been concentrated by a factor of 7.3 X andto have an infectivity titer of 7.0.

Example 6 Mumps virus, Habel strain, grown in embryonated eggs andinactivated with formalin, was concentrated and purified according tothe procedure of Example 1. The inactivated vaccine originally had ahemagglutination titer of 1:512; after being treated, the concentrationof the virus was 10X, and a 1:10 dilution had a titer of 1:512. Theinactivated vaccine originally contained 6 mg. of protein permilliliter; after being treated, the 10 X concentrate contained 10.42mg. per milliliter.

Example 7 Live measles virus, grown in primary chick embryo cells, wassuccessfully concentrated and purified according to the procedure andessentially under the conditions of Example 1. The original virus had aninfectivity titer of 2.63, while the treated virus, having a 10Xconcentration, had a titer of 3.44.

Example 8 Murivirus, grown in Primary Green Monkey kidney tissueculture, was successfully concentrated and purified according to theprocedure and essentially under the conditions of Example 1. Theoriginal virus had an infect-ivity titer of 5.2; after treatment, it hada concentration of 6x and an infectivity titer of 5.62.

While the invention has been described by reference to certain specificmaterials, operations, and conditions, it is to be understood thatnumerous modifications and variants thereof will occur to those skilledin the art which do not depart from the spirit of the invention. Suchmodifications and variants are to be considered as lying within thescope of the invention, as defined by the following claims.

-I claim:

1. In a method for the purification and concentration of an impure viruspreparation containing proteina-ceous impurities, which method includesthe steps of contacting said impure virus preparation in an aqueousmedium with in situ precipitated calcium phosphate under conditionsefiective to selectively adsorb said virus thereon while substantiallyrejecting the impurities, and recovering said virus from the resultingadsorbate in substantially purified and concentrated 'form, the improvedmethod for said recovery which comprises contacting said adsorbate withan aqueous solution of ethylenediaminetetraacetic acid of pH 7.8433,whereby the calcium phosconverted into a complex with saidethylenediaminetetraacetic acid and is dispersed in said solution, andwhereby said virus is released from said adsorbate.

2. The method of claim 1 wherein said impure virus preparation is aninactivated polyvalent poliomyelitis vaccine.

3. The method of claim 1 wherein said impure virus preparation is anattenuated polyvalent poliomyelitis vaccine.

4. The method of claim 1 wherein said impure virus preparation is aninactivated influenza. vaccine.

5. The method of claim 1 wherein said impure virus preparation is aninactivated measles vaccine.

6. The method of claim 1 wherein said impure virus preparation is anattenuated measles vaccine.

7. In a method for the purification and concentration of an impure viruspreparation containing proteinaceous impurities, which method includesthe steps of contacting said impure virus preparation in an aqueousmedium with in situ precipitated calcium phosphate under conditionseffective to selectively adsorb said virus thereon while substantiallyrejecting the impurities, and recovering said virus from the resultingadsorbate in substantially purified and concentrated form, the improvedmethod for said recovery which comprises contacting said adsorbate withan aqueous solution of ethylenediaminetetraacetic acid of pH 8.0-8.2,whereby the calcium phosphate in said adsorbate is converted into acomplex with said ethylenediaminetetraacetic acid and is dispersed insaid solution, and whereby said virus is released from said adsorbate.

8. In a method for the purification and concentration of an impure viruspreparation containing proteinaceous impurities, which method includesthe steps of contacting said impure virus preparation in an aqueousmedium with in situ precipitated calcium phosphate under conditionsefiective to selectively adsorb said virus thereon while substantiallyrejecting the impurities, and recovering said virus from the resultingadsorbate in substantially purified and concentrated form, the improvedmethod for said recovery which comprises commingling said adsorbate withan aqueous solution of ethylenediarninetetraacetic acid of pH 7.8-8.3,whereby the calcium phosphate in said adsorbate is converted into acomplex with said ethylenediaminetetraacetic acid and is dispersed insaid solution, and whereby said virus is released from said adsorbate,and dialyzing said complex at least in part from said solution.

9. In a method for the purification and concentration of an impure viruspreparation containing proteinaceous impurities, which method includesthe steps of contacting said impure virus preparation in an aqueousmedium with in situ precipitated calcium phosphate under conditionseffective to selectively adsorb said virus thereon while substantiallyrejecting the impurities, and recovering said virus from the resultingadsorbate in substantially purified and concentrated form, the improvedmethod for said recovery which comprises commingling said adsorbate withan aqueous solution of ethylenediaminetetraacetic acid of pH 7.8-8.3,whereby the calcium phosp'hate in said adsorbate is converted into acomplex with said ethylenediaminetetraacetic acid and is dispersed insaid solution, and whereby said virus is released from said adsorbate,and dialyzing said complex at least in part from said solution withaqueous glycine-sodium chloride solution.

10. In a method for the purification and concentration of an impurevirus preparation containing proteinaceous impurities, which methodincludes the steps of contacting said impure virus preparation in anaqueous medium with in situ precipitated calcium phosphate underconditions effective to selectively adsorb said virus thereon whilesubstantially rejecting the impurities, and recovering said virus fromthe resulting adsorbate in substantially purified and concentrated form,the improved method for said recovery which comprises commingling saidadsorbate with an aqueous solution of ethylenediaminetetraacetic acid ofpH 7.8-8.3, wherby the calcium phosphate in said adsorbate is convertedinto a complex with said ethyleneiiaminetetraacetic acid and isdispersed in said solution, and whereby said virus is released from saidadsorbate, adding to the resulting solution a stabilizing proportion ofpolysorbitan monooleate, and dialyzing said complex at least in partfrom said solution with aqueous glycinesodium chloride solutioncontaining a stabilizing proportion of polysorbitan monooleate.

References Qited by the Examiner UNITED STATES PATENTS 3,132,073 5/1964MacFarlane et al. 167-78 5 LEWIS GOTTS, Primary Examiner.

RICHARD L. HUFF, Assistant Examiner.

1. IN A METHOD FOR THE PURIFICATION AND CONCENTRATION OF AN IMPURE VIRUSPREPARATION CONTAINING PROTEINACEOUS IMPURITIES, WHICH METHOD INCLUDESTHE STEPS OF CONTACTING SAID IMPURE VIRUS PREPARATION IN AN AQUEOUSMEDIUM WITH IN SITU PRECIPITATED CALCIUM PHOSPHATE UNDER CONDITIONSEFFECTIVE TO SELECTIVELY ABSORB SAID VIRUS THEREON WHILE SUBSTANTIALLYREJECTING THE IMPURTIES, AND RECOVERING SAID VIRUS FROM THE RESULTINGABSORBATE IN SUBSTANTIALLY PURIFIED AND CONCENTRATED FORM, THE IMPROVEDMETHOD FOR SAID RECOVERY WHICH COMPRISES CONTACTING SAID ADSORBATE WITHAN AQUEOUS SOLUTION OF ETHYLENEDIAMINETETRAACETIC ACID OF PH 7.8-8.3,WHEREBY THE CALCIUM PHOSPHATE IN SAID ABSORBATE IS CONVERTED INTO ACOMPLEX WITH SAID ETHYLENEDIAMINETETRAACETIC ACID AND IS DISPERSED INSAID SOLUTION, AND WHEREBY SAID VIRUS IS RELEASED FROM SAID ADSORBATE.